SPADA Meeting Book

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4.3.11 Multiplexing is a complex system

Multiplex PCR is a complex system with many interacting variables (e.g., multiple primers, 516 genomic DNA from the target(s) of interest and contaminating genomes from the sample 517 matrix). Optimizing one set of primers can result in an unexpected interaction with another set of 518 primers in the reaction to create unexpected false amplicons from the exclusivity and background 519 databases or primer-amplicon cross-hybridization reactions. In addition, as the size of the 520 multiplexed reaction grows larger, there is a combinatorial explosion in the permutations of 521 possible multiplex reactions. Consider a 20-plex reaction with 10 primer design candidates (i.e. 522 the top scoring singleplex designs) for each of the 20 targets: the number of possible multiplex 523 reactions to consider is 10 20 . An iterative empirical approach can only assess a few reactions 524 (without knowledge of the interacting variables) and thus samples a very limited amount of the 525 sequence design space, almost always resulting in a sub-optimal design and often complete 526 failure. Even an iterative brute-force computational approach using high-performance computing 527 that could test millions of combinations of multiplex sets will vastly under sample the number of 528 possible reactions. For the 20-plex example with 10 candidate solutions, it would be 529 computationally intractable to run thermodynamic scanning (e.g., PrimerBLAST or 530 ThermoBLAST ) for all 10 20 possible multiplex permutations to ensure that false positives are 531 minimized. Thus, a modern sophisticated algorithm is needed to solve the combinatorial 532 explosion thoroughly and find the optimum multiplex design. The program PanelPlex uses a 533 depth-first search with a pruning algorithm to accomplish the multiplexing design task and 534 guarantees finding the optimum solution (or top N solutions) out of the entire space of possible 535 multiplexes. We recommend using such multiplex optimization algorithms to greatly reduce the 536

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